Membrane-bound ribosomes form Bacillus stearothermophilus contain an extra protein (M-protein) not found in free ribosomes. In vitro binding studies will be employed to determine if the M-protein is an essential requirement for the ribosome-membrane interaction. The possibility that the M-protein plays a role in the vectorial transport of puromycin-released protein across the membrane will also be investigated. A bifunctional cross-linking reagent, methyl 4- mercaptobutyrimidate, will be utilized to define both the ribosomal and membrane protein neighborhoods in close proximity to the M-protein. The formation of membrane-bound polyribosomes will be traced by pulse-labeling nascent polypeptides in the presence of a selective inhibitor of membrane-associated protein synthesis, followed by a "chase" in the absence of the inhibitor. The hypothesis that an extracellular enzyme, alpha-amylase, is preferentially synthesized on membrane-bound polyribosomes will be tested. Nascent polypeptide chains synthesized in vitro on these polyribosomes as well as on polyribosomes not attached to membranes will be assayed for amylase-like protein both immunologically and chemically. If such site-specific synthesis can be demonstrated, membrane-bound and free ribosomes will be examined for the presence of messenger-discriminating species of initiation factor IF3. Mutants defective in the secretion of amylase will be isolated and attempts will be made to determine the locus responsible for this lesion.